Growth characteristics of HCT116 xenografts lacking asparagine synthetase vary according to sex

Background Sex-related differences in colorectal (CRC) incidence and mortality are well-documented. However, the impact of sex on metabolic pathways that drive cancer growth is not well understood. High expression of asparagine synthetase (ASNS) is associated with inferior survival for female CRC patients only. Here, we used a CRISPR/Cas9 technology to generate HCT116 ASNS−/− and HCT 116 ASNS+/+ cancer cell lines. We examine the effects of ASNS deletion on tumor growth and the subsequent rewiring of metabolic pathways in male and female Rag2/IL2RG mice. Results ASNS loss reduces cancer burden in male and female tumor-bearing mice (40% reduction, q < 0.05), triggers metabolic reprogramming including gluconeogenesis, but confers a survival improvement (30 days median survival, q < 0.05) in female tumor-bearing mice alone. Transcriptomic analyses revealed upregulation of G-protein coupled estrogen receptor (GPER1) in tumors from male and female mice with HCT116 ASNS−/− xenograft. Estradiol activates GPER1 in vitro in the presence of ASNS and suppresses tumor growth. Conclusions Our study indicates that inferior survival for female CRC patients with high ASNS may be due to metabolic reprogramming that sustains tumor growth. These findings have translational relevance as ASNS/GPER1 signaling could be a future therapeutic target to improve the survival of female CRC patients. Supplementary Information The online version contains supplementary material available at 10.1186/s40246-024-00635-3.

RNA Seq Library Prep: mRNA was purified from approximately 200ng of total RNA with oligo-dT beads and sheared by incubation at 94C in the presence of Mg (Roche Kapa mRNA Hyper Prep Cat# KR1352, Basel, Switzerland).Following first-strand synthesis with random primers, second strand synthesis and A-tailing was performed with dUTP for generating strandspecific sequencing libraries.Adapter ligation with 3' dTMP overhangs were ligated to library insert fragments.Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends.Strands marked with dUTP were not amplified.Indexed libraries are quantified by qRT-PCR using a commercially available kit (Roche KAPA Biosystems Cat# KK4854, Basel, Switzerland) and insert size distribution determined by the Agilent Bioanalyzer.Samples with a yield of ≥ 0.5 ng/ul and a size distribution of 150-300bp are used for sequencing.
Flow Cell Preparation and Sequencing: Sample concentrations were normalized to 1.2 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yields 25 million passing filter clusters per sample.Samples were sequenced using 100bp paired-end sequencing on an Illumina NovaSeq6000 according to Illumina protocols.The 10bp unique dual index is read during additional sequencing reads that automatically follow the completion of read 1.Data generated during sequencing runs were simultaneously transferred to the YCGA high-performance computing cluster.A positive control (prepared bacteriophage Phi X library) provided by Illumina was spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time.
Pre-processing of RNA-Seq Data and Storage: Signal intensities were converted to individual base calls during a run using the system's Real Time Analysis (RTA) software.Base calls were transferred from the machine's dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis.Primary analysissample de-multiplexing and alignment to the human genome -was performed using Illumina's CASAVA 1.8.2 software suite.The error rate was less than 2% and the distribution of reads per sample in a lane was within reasonable tolerance.

Figure S1 :
Figure S1: Dose response effects of L-Asparaginase (L-Asp) treatment on HCT116 ASNS +/+ and HCT116 ASNS -/-cell lines.(A) L-Asp reduces spheroid formation in the HCT116 ASNS lines.Cells were cultured in RPMI with sufficient asparagine and supplemented with 4 mM glutamine to scale up cells before asparaginase treatment.Representative images of spheroids cultured for 7 days after seeding using 4x objective lens in an inverted microscope.0, 0.5, 2 and 4 IU/mL L-asp provided.(B) Quantitation of L-Asp treatment experiment (C) Metabolomic analysis showing asparagine levels in HCT116 ASNS +/+ and HCT116 ASNS -/-cell lines.Cells were cultured in RPMI and supplemented with 4 mM glutamine.Individual dot plot represents mean ± SEM with two-way ANOVA used to determine significance in spheroid growth in HCT116 ASNS +/+ and HCT116 ASNS -/-cells treated with L-Asp (FigS1B).*denotes q < 0.05 and*** denotes q < 0.0001 using Benjamini Hochberg FDR correction method for multiple comparisons.

Figure S4 :
Figure S4: Network analysis of metabolites detected in HCT116 ASNS tumor xenograft (female mice, study 2).Network analysis was conducted with Ingenuity Pathway Analysis software on 36 tumor metabolites that were significantly altered in the HCT116 ASNS -/-female tumors (n=8) compared to HCT116 ASNS +/+ female tumors (n=10) from study 2. Significantly increased metabolites are highlighted in red.Network score = 33.The score represents the negative exponent of the right-tailed Fisher's exact test.Statistical significance was determined using q < 0.05.

Figure S5 :Figure S6 :
Figure S5: Network analysis of significantly altered genes using IPA (Study 1).ERK and TNF signaling were predicted to be activated by ASNS deletion in the female tumor bearing R2G2 mice.Network analysis using the top differentially expressed genes from RNA-Seq data in study 1.Molecules in green indicate significantly downregulated genes and molecules in red indicate significantly upregulation. .Network score = 45.The score represents the negative exponent of the right-tailed Fisher's exact test p-value